The Erythroxylaceae of southern Africa : a metabolomical, genetic and chemotaxonomical evaluation

The Erythroxylaceae family consists of trees and shrubs sub-divided into four genera, some of which can produce highly valued, or precursors to, medicinal compounds, including atropine, cocaine, tropacocaine and tigloidine, amongst others. Selected species within the Erythroxylum and Nectaropetalum genera are endemic to southern Africa, showing great pharmaceutical potential based on literature reports and their traditional uses. Discrepancies from specific reports regarding the presence of tropane alkaloids initiated a GC-MS-based phytochemical investigation of E. delagoense, E. emarginatum and E. pictum. Results from these investigations showed the presence of atropine, tropacocaine, and the precursors, tropinone and methylecgonine, in selected South African Erythroxylum species, providing clarity to these discrepancies. Moreover, the species with the most pharmaceutical potential based on its tropane alkaloid content, E. emarginatum, was further investigated using plant tissue culture-based tropane alkaloid precursor feeding strategies to determine the potential of upregulating these tropanes in vitro. These experiments, conducted on leaf callus cultures of E. emarginatum revealed the upregulation of tropine, a precursor to tropacocaine and atropine, when tropinone was introduced into the culture media. Morphologically differentiating between the three Erythroxylum species and the two Nectaropetalum species (N. capense and N. zuluense) has proven troublesome. Thus, alternative chemical- (NMR and GC-MS) and molecular (DNA barcoding)-based identification strategies were investigated. These analyses provided species-level resolution on a chemical and molecular basis. The shared morphological characteristics, phytochemical profiles and phylogenetic relatedness to Neotropical cocaine-producing varieties, initiated investigations into the final steps of cocaine biosynthesis in these Old-World species. Results from the comparative Sanger sequencing analyses and enzyme-ligand docking simulations revealed the presence of a highly mutated cocaine synthase gene, the penultimate cocaine biosynthesis enzyme, in all five investigated species. This provided evidence for their inability to biosynthesise cocaine and the first evidence of an evolutionary-related cocaine synthase gene from these Old-World species. The collective results from this PhD thesis emphasise the pharmaceutical potential of the South African coca plants while broadening our understanding of the effect of biosynthetic pathway evolution.